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Detection of blaTEM and blaCTX-M genes by multiplex polymerase chain reaction amongst uropathogenic Escherichia coli strains isolated from hospitalized patients in Kolkata, India

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Research Paper 01/12/2011
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Detection of blaTEM and blaCTX-M genes by multiplex polymerase chain reaction amongst uropathogenic Escherichia coli strains isolated from hospitalized patients in Kolkata, India

Mandira Mukherjee, Shreya Basu, Monalisa Majumdar
Int. J. Biosci.1( 6), 64-69, December 2011.
Certificate: IJB 2011 [Generate Certificate]
Key: ESBLMultiplex PCRUPEC

Abstract

Production of extended-spectrum β-lactamase (ESBL) is one of the most important resistance mechanisms that hamper the antimicrobial treatment of infections caused by uropathogenic Escherichia coli (UPEC). The objective of the study was to determine the blaTEM and blaCTX-M producers simultaneously in a single tube reaction by multiplex PCR technique using gene specific primer sets. A total of 100 E. coli strains, isolated from hospitalized patients were phenotypically screened for ESBL production using drug-inhibitor combination disks, namely, ceftazidime-clavulanate (30+10μg) and cefotaxime-clavulanate (30+10μg) following CLSI guidelines. Out of the phenotypically screened 85 ESBL producers, multiplex PCR carried out on plasmid DNA alone indicated 50.5 % and 55.3% positivity for blaCTX-M and blaTEM gene respectively, whereas PCR on both plasmid and genomic DNA showed 81.1 % positivity for blaCTX-M and 83.5% positivity for blaTEM genes. A high percentage (52.9%) of the isolates was found to harbor both genes simultaneously. Moreover amplification of both plasmid and genomic DNA from the non-ESBL producers indicated that 6 of the isolates harbored blaTEM gene. Amplification of whole genomic DNA increased the positivity of detection, compared to amplification of plasmid DNA alone, suggesting β-lactamase expression was controlled by both chromosomal and plasmid DNA. This study for the first time reports the prevalence of the two most common ESBL responsive genes using multiplex PCR in hospitalized patients from Kolkata, India. This technique provided an efficient and rapid differentiation of ESBLs and could be used as a rapid tool for epidemiological studies.

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